Developing a Rat Analogue of a Human Transglutaminase 2 Inhibitory Antibody to Facilitate Preclinical Testing in Treating Kidney Fibrosis

纤维化或瘢痕形成是许多相对常见的慢性疾病的常见特征。这些疾病包括多种类型的肺病、肝病和心脏病，以及基本上所有形式的慢性肾病。鉴于目前还没有公认的抗瘢痕形成/抗纤维化治疗方法可用于每年最终导致数百万人死亡的这种疾病，对瘢痕形成过程背后机制的研究是一个活跃的领域。因此，开发能够阻断或减少疤痕影响的药物所提供的潜在治疗益处是相当可观的。来自一系列不同小组和实验设计的累积数据强调了组织转谷氨酰胺酶（转谷氨酰胺酶2型，TG2）是瘢痕形成过程的关键效应因子。谷氨酰胺转胺酶是一个密切相关的酶家族，它们都通过共价的ε （γ -谷氨酰）赖氨酸键催化肽链的交联。正是TG2的这种肽交联活性在慢性瘢痕形成过程中产生了细胞外基质沉积的增加和稳定。在慢性肾脏疾病（CKD）的动物模型中，无论是通过遗传还是通过使用小分子抑制剂，TG2的敲低已被证明具有明显的有益作用。在实验的时间尺度上，疤痕和纤维化减少，肾功能保存高达80%。将这些结果转化为患者环境将在CKD领域以及其他以慢性瘢痕形成为特征的疾病领域具有深远的治疗应用。延长肾功能和延迟透析或肾移植的需要将大大提高慢性肾病患者的生活质量，并对卫生服务产生重大的成本影响。鉴于TG2抑制剂开发的明确案例，这一研究领域有很大的兴趣。抑制剂的开发受到TG家族8个不同成员之间结构保守程度的阻碍。催化位点或“核心”是高度保守的，所有现有的小分子候选物都表现出与其他相关酶的交叉反应性。这对于维持真皮层完整性至关重要的TG1和TG3以及凝血过程的主要成分因子XIIIa尤为重要。抑制这些相关的TG分子会产生严重的副作用，使现有的小分子抑制剂无法发挥治疗作用。由于抗体靶向的非常精确的性质，单克隆抗体方法提供了克服这些特异性问题的潜力。利用一种创新的免疫方法，我们成功地产生了一系列tg2特异性抑制单克隆抗体。所有这些都是人类TG2特异性的，并且与酶家族的其他成员没有交叉反应性。我们已经在不同的体外系统中建立了这些单克隆抗体的抑制潜力，并获得了IC50数据。这些抗体中最有效的已被人源化，具有亲和力和抑制活性被保留。这种潜在疗法的下一阶段是在FDA批准的慢性纤维化动物模型中产生临床前数据。最有效的单克隆，AB1和其他候选，都是对人TG2特异性的，对小鼠或大鼠TG2缺乏明显的抑制作用。因此，该项目的目的是使我们能够产生针对大鼠和小鼠TG2的同源抑制抗体，以便我们能够收集这一关键的临床前数据，吸引下游商业合作伙伴，并加速这一潜在的新型治疗药物的开发。

Fibrosis, or scarring, is a frequent feature of a number of relatively common, usually chronic, diseases. These include many types of lung, liver and heart disease and essentially all forms of chronic kidney disease. Studies of the mechanisms underlying the scarring process is an area of intense activity given that currently there is no recognized anti-scarring / anti-fibrotic therapy available for a condition that is ultimately fatal for millions of people each year. Subsequently the potential therapeutic benefits offered by the development of agents able to block or reduce the impact of scarring are considerable. Cumulative data from a range of different groups and experimental designs have highlighted tissue transglutaminase (transglutaminase type 2 ,TG2) as a key effector of the scarring process. Transglutaminases are a family of closely related enzymes that all catalyze the crosslinking of peptide chains via covalent epsilon (gamma-glutamyl) lysine bonds. It is this peptide crosslinking activity of TG2 that acts to produce the increased deposition and stabilization of extracellular matrix observed in chronic scarring. Knockdown of TG2, either genetically, or through the use of small molecule inhibitors, has been shown to have clear beneficial effects in animal models of chronic kidney disease (CKD). Scarring & fibrosis is reduced and kidney function preserved by up to 80% over the timescale of the experiments. Translation of these results into the patient setting would have profound therapeutic application in the area of CKD, and possibly other diseases that feature chronic scarring. Prolonging kidney function and delaying the requirement for dialysis or renal transplantation would offer a substantial increase in the quality of life of CKD patients and have major cost implications for the health service. Given the clear case for the development of TG2 inhibitors, there is much interest in this area of research. Development of inhibitors have been hampered by the degree of structural conservation between the 8 different members of the TG family. The catalytic site, or 'core', is highly conserved and all existing small molecule candidates have exhibited prohibitive levels of cross reactivity with other related enzymes. This is particularly important in the case of TG1 and TG3, which are critical to maintaining integrity of the dermis, and factor XIIIa, a major component of the clotting process. Inhibition of these related TG molecules produces serious side effects that would preclude a therapeutic role for existing small molecule inhibitors. A monoclonal antibody approach to this problem offers the potential to overcome these specificity issues due to the very precise nature of antibody targeting.Using an innovative immunization approach we have successfully generated a series of TG2-specific inhibitory monoclonal antibodies. All are specific for human TG2 and show no cross reactivity to other members of the enzyme family. We have established the inhibitory potential of these mAbs in different in vitro systems and derived IC50 data. The most effective of these antibodies has been humanised, with the affinity and inhibitory activity being preserved. The next stage in the development of this potential therapeutic is to generate preclinical data in FDA approved animal models of chronic fibrosis. The most effective monoclonal, AB1, and the other candidates, are all specific for human TG2, and lack significant inhibitory action against mouse or rat TG2. Accordingly, the aim of this project is to allow us to generate orthologous inhibitory antibodies against rat and mouse TG2, in order that we can gather this crucial preclinical data, attract downstream commercial partners, and accelerate the development of this potentially new class of therapeutic agents.